We have constructed an oncolytic adenovirus vector named "KD3" that replicates selectively in cancer cells as opposed to normal cells because of a mutation in the adenovirus E1A gene that prevents E1A proteins from deregulating the cell cycle in normal cells. KD3 was designed to overexpress the E3-11.6K protein (Adenovirus Death Protein, ADP). Overexpression of ADP allows for efficient egress of KD3 from infected cancer cells and the spread of KD3 from cell-to-cell. We also have constructed and partially characterized in cell culture a replication-defective vector named "Tet-On-TRAIL" that expresses TNFrelated apoptosis-inducing ligand (TRAIL) under the control of a tetracycline-regulatable cassette. TRAIL is a member of the TNF family of death ligands that has been shown to induce apoptosis in a majority of cancer cells but not in most normal cells. When cells are co-infected with Tet-On-TRAIL and KD3, KD3 complements the replication and spread of Tet-On-TRAIL. When the Tet promoter is induced by doxyclycline (DOX), Tet-On-TRAIL synthesizes TRAIL very abundantly. We propose to characterize this "binary" vector system in cell culture and xenografts in nude mice with the long-term goal of obtaining preclinical data for a clinical trial. Tumors should be destroyed by two independent mechanisms, the replication and cell-to-cell spread of KD3 plus Tet-On-TRAIL, and the apoptosis-inducing ability of TRAIL on uninfected cancer cells. The vector system should be restricted to tumors by virtue of the E 1A mutation in KD3 and the cancer cell specificity of TRAIL. In cell culture experiments, we will examine TRAIL protein by immunoblot and immunofluorescence and TRAIL function by apoptosis assays. In order to determine if there is a bystander effect, cells infected with Tet-On-TRAIL+KD3+DOX will be mixed with uninfected cells and the death of the latter will be assayed. In vivo, we will inject Tet-On-TRAIL+KD3+DOX plus control vectors into subcutaneous human Hep3B liver cancer tumors in nude mice and examine the growth and apoptosis of the tumors, including uninfected contralateral tumors which may be destroyed by TRAIL released from the infected tumors. The possible toxicity to the mouse liver will be studied.